PROJECT SUMMARY/ABSTRACT The goal of this exploratory study is to develop and validate a novel genomewide screening technology for identifying noncoding RNAs (ncRNAs) that may serve as diagnostic and/or prognostic biomarkers for cancer chemoresistance to targeted therapies. Only <2% of human genome is transcribed into mRNA, leaving most of the transcribed human genome encoding RNA with little known functions. The past several years have witnessed a huge expansion of our knowledge about the important functions of ncRNAs, such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), in many physiological and pathologic processes including cancer drug resistance. However, current identification of ncRNAs primarily relies on large-scale deep sequencing and computational predictions. Deep sequencing is limited by the low expression level of ncRNAs, while computational predictions are challenged by the presence of preserved secondary structural elements within ncRNAs (esp. lncRNAs) without sequence conservation. It?s an even more daunting challenge to functionally validate the putative ncRNAs. Thus, there is an urgent need to develop innovative technology to identify and annotate functional ncRNAs. We have recently constructed a high-content and completely-randomized 19-nt short RNA (sRNA) library or 19nRNA library, which expresses ~2.4x107 unique sequences driven by the converging U6 and H1 promoters. In our pilot study, we introduced the 19nRNA library into human melanoma line A375 cells and successfully established stable BRAF and MEK inhibitor- resistant lines that exhibit similar characteristics of melanoma cells with naturally-acquired resistance to BRAFi/MEKi. Furthermore, the enriched 19nRNA sequences retrieved through next-generation sequencing were shown to confer robust resistance to BRAFi in nave melanoma cells, and rarely exactly match protein- coding genes but mapping to ncRNAs, suggesting that the 19nRNA library may be a novel and effective technology to identify functional ncRNAs. Here, we use human melanoma cells as a model system and propose to fully validate the technologic and biological reproducibility of the 19nRNA screening technology by focusing on three aims. Aim #1 is to determine the technological reproducibility of the 19nRNA screening technology. Aim #2 is to determine the biological reproducibility of the 19nRNA screening technology. Aim #3 is to functionally validate of the 19nRNA screening technology. We believe the proposed 19nRNA screening technology is innovative and paradigm-shifting. A successful completion of the proposed work should validate the 19nRNA screening technology as a novel and powerful tool to unravel the pervasive yet elusive regulatory functions of ncRNAs in cellular processes and the development and/or progression of human diseases, such as tumorigenesis and chemoresistance. Ultimately, ncRNAs identified from such functional screening may serve as important diagnostic/prognostic biomarkers of chemoresistance, novel drug targets, and/or ncRNA-based therapeutics so to advance our agenda for personalized precision medicine.